Targeted at learning about pathogenic processes of inflammatory eye diseases, this project focused in FY 2011 mainly on populations of T-cells involved in these processes. Specifically, we extended our investigation of two populations of recently identified T-helper cells, specifically producing IL-17 ("Th17" cells), or IL-9 ("Th9" cells). In addition, we analyzed (i) the role of the SLAT/Def6 gene in immune-mediated ocular inflammation and (ii) the capacity of the newly developed non-toxic endogenous aryl hydrocarbon receptor ligand, ITE, to inhibit development of experimental autoimmune uveitis (EAU) and immune responses against the uveitogenic antigen. 1) Th17 Cells. We extended our studies aimed at further analysis of the immunobiological and pathogenic features of the two subpopulations of Th17 cells. In previous studies, detailed in our FY 2009 and FY 2010 Reports, we described new procedures we developed for the generation of two subpopulations of Th17 cells, of which one is pathogenic the other is not. The experimental system we use consists of two lines of transgenic mice. T-cells of one line, designated 3A9, express T-cell receptor (TCR) specific against hen egg lysozyme (HEL), while mice of the other line, named HEL-Tg, express HEL in their eyes. When acquiring pathogenicity, T-cells of the 3A9 line induce inflammation in eyes of the HEL-Tg recipients. The pathogenic subpopulation of Th17 cells is generated by activation of naive CD4 lymphocytes of 3A9 mice with HEL and antigen-presenting cells (APC), whereas the non-pathogenic subpopulation is generated by activation of the naive CD4 lymphocytes by antibodies against the surface molecules CD3 and CD28. Both subpopulations are polarized by incubation with the cytokines IL-6 and TGF-beta. This study was extended in FY 2011 by introducing a new experimental system to examine the hypothesis that naive CD4 acquire the pathogenic Th17 phenotype by activation via cell-cell interaction with APC which present the target antigen. The new system consists of two culture compartments separated by semi-permeable membrane. One compartment harbors naive CD4 cells activated by plate-bound anti-CD3/CD28 antibodies;the other harbors APC with the target antigen, along with naive CD4 cells. Following incubation, the CD4 in the latter compartment developed the pathogenic Th17 phenotype, whereas those in the former did not. These observations thus indicate that, unexpectedly, development of pathogenic capacity by Th17 cells requires cell-cell interaction with APC presenting the target antigen. Interestingly, unlike Th17 cells, Th1 cells acquire pathogenicity when activated by either anti-CD3/CD28 antibodies or APC/antigen;when tested by the system detailed above, pathogenic Th1 were generated at either culture compartment. Further investigation is needed to analyze this surprising major difference between Th1 and Th17. 2. Th9 cells. Our studies in FY 2011 yielded new information concerning the population of Th9 cells. These cells are mainly involved in allergic reactions and immune responses against helminthes. Our previous studies revealed several unique features of the Th9 cells, including their early production of IL-9 and, in particular, their rapid secretion of this signature cytokine. It is conceivable these features are related to the physiological functions of Th9. Our lines of Th9 cells were generated by the target antigen, presented by APC. Th9 lines in the majority of published studies, however, were generated by activating the naive CD4 cells with antibodies against CD3 and CD28, a commonly used procedure for activation of naive lymphocytes. In view of our observations showing differences between Th17 lines generated by these two procedures, we compared several basic features of Th9 generated by antigen/APC or anti-CD3/CD28 antibodies. The two types of lines similarly produced IL-9, as well IL-10, another cytokine that is typically produced by Th9 cells (Tan et al., J. Immunol., 185:6795, 2010). Similarities were observed in the kinetics and levels of the two cytokines. Remarkable differences were found, however, between Th9 lines generated by the two procedures in their immunopathogenic capacity and homing to and proliferation in the recipient spleen. Th9 generated by antigen/APC were found to acquire pathogenicity only when tested after 3 days of culture;no pathogenicity was detected in cells of the same cultures when collected one day later, i.e., day 4 in culture. In contrast, Th9 generated by anti-CD3/CD28 antibodies retained their pathogenic capacity for at least 6 days in culture. Adoptively transferred activated T-helper cells migrate into the recipient spleen, where they multiply before exiting to the periphery, where the target antigen is located. Exposure of the migrating lymphocytes to the antigen initiates the pathogenic process, ocular inflammation in our system. A good correlation was found in our study between the proliferation in the recipient spleen and the pathogenic capacity of Th9 cells. Thus, Th9 cells generated by antigen/APC multiplied in the recipient spleen only when harvested after 3 days in culture, but lost this capacity when harvested on day 4, in line with their pathogenic capacity. Th9 generated by anti-CD3/CD28 antibodies, on the other hand, proliferated in the recipient spleen and induced inflammation when harvested on all tested days in culture. 3) Participation of the SLAT/Def6 gene in immune-mediated ocular inflammation. This study was initiated in FY 2010, but carried out mostly in FY 2011. Mice deficient in the SLAT/Def6 gene were compared with wild-type controls for their capacity to develop EAU and immune responses against the uveitogenic antigen, interphotoreceptor retinoid-binding protein (IRBP). The deficient mice were inferior to their wild-type controls by all the aforementioned parameters, thus providing evidence to show that the SLAT/Def6 gene plays a major role in development of the immune response responsible for EAU induction. 4) Immunosuppressive capacities of ITE. The development of Th17 and T-regulatory (Treg) cells is controlled by the ligand-activated transcription factor aryl hydrocarbon receptor (AHR). An AHR ligand that has been well investigated, TCDD, or Dioxin, is highly toxic and cannot be used in therapy. More recently, a non-toxic AHR ligand was developed and designated "ITE" 2-(1H-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester. ITE was found to effectively inhibit experimental autoimmune encephalomyelitis (EAE). We examined the effects of ITE on development of EAU and found it to be highly inhibitory in three strains of mice, B10.A, B10RIII and C57Bl/6. To further analyze the ITE immunosuppressive activity, we tested its effect on immune responses known to play major roles in EAU induction. Treatment with ITE inhibited the development of cellular immune response, assessed by the proliferative response of lymphocytes to the immunizing/uveitogenic antigen and the production of the pro-inflammatory cytokines, IL-17 and interferon-gamma. Importantly, the non-toxicity of ITE indicates this compound could be considered for therapy of pathogenic autoimmune conditions.